EP1934245B1 - Agonistes selectifs du recepteur y2 pour applications therapeutiques - Google Patents

Agonistes selectifs du recepteur y2 pour applications therapeutiques Download PDF

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EP1934245B1
EP1934245B1 EP05794808A EP05794808A EP1934245B1 EP 1934245 B1 EP1934245 B1 EP 1934245B1 EP 05794808 A EP05794808 A EP 05794808A EP 05794808 A EP05794808 A EP 05794808A EP 1934245 B1 EP1934245 B1 EP 1934245B1
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peptide
receptor
iii
amino acid
gln34
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EP1934245A1 (fr
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Thue Schwartz
Feng Wang
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7TM Pharma AS
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    • C07K14/57545—Neuropeptide Y
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Definitions

  • the invention relates to peptide or peptidic compounds that act as selective agonists of the Y2 relative to the Y1 and Y4 receptors, and to their use in treatment of conditions responsive to activation of Y2 receptors, for example in treatment of obesity and overweight, and conditions in which these are considered contributory factors, and for induction of angiogenesis.
  • the PP-fold family of peptides - NPY (Neuropeptide Y) (human sequence - SEQ ID. No:1), PYY (Peptide YY) (human sequence- SEQ ID. No:2), and PP (Pancreatic Polypeptide) (human sequence - SEQ ID. No:3), are naturally secreted homologous, 36 amino acid, C-terminally amidated peptides, which are characterized by a common three-dimensional, structure - the PP-fold - which is surprisingly stable even in dilute aqueous solution and is important for the receptor recognition of the peptides.
  • NPY is a very wide-spread neuropeptide with multiple actions in various parts of both the central and peripheral nervous system acting through a number of different receptor subtypes in man: Y1, Y2 ; Y4 and Y5.
  • the main NPY receptors are the Y1 receptor, which generally is the post-synaptic receptor conveying the "action" of the NPY neurones and the Y2 receptor which generally is a pre-synaptic, inhibitory receptor.
  • NPY neurones - which also express the melanocortin receptor antagonist / inverse agonist AgRP (agouti related peptide) - act as the primary "sensory” neurones in the stimulatory branch of the arcuate nucleus.
  • the NPY/AgRP neurones together with the inhibitory POMC/CART neurones monitor the hormonal and nutritional status of the body as these neurones are the target for both the long-term regulators such as leptin and insulin and short term regulators such as ghrelin and PYY (see below).
  • the stimulatory NPY/AgRP neurones project for example to the paraventricular nucleus - also of the hypothalamus - where its postsynaptic target receptors are believed to be Y1 and Y5 receptors.
  • NPY is the most potent compound known in respect of increasing food intake, as rodents upon intracerebroventricular (ICV) injection of NPY will eat until they literally burst.
  • AgRP from the NPY/AgRP neurones acts as an antagonist mainly on melanocortin receptors type 4 (MC-4) and block the action of POMC derived peptides - mainly aMSH - on this receptor.
  • the action of AgRP is - just like the NPY action - a stimulatory signal for food intake (i.e. an inhibition of an inhibition).
  • inhibitory - pre-synaptic - Y2 receptors which are the target both of locally released NPY as well as a target for the gut hormone PYY - another PP-fold peptide.
  • PYY is released during a meal - in proportion to the calorie content of the meal - from entero-endocrine cells in the distal small intestine and the colon, to act both in the periphery on Gl-tract functions and centrally as a satiety signal.
  • PYY is believed to function as an inhibitor- an "illeal break" - on for example-upper Gl-tract motility, gastric acid and exocrine pancreatic secretion.
  • PYY is believed to act mainly on the presynaptic, inhibitory Y2 receptors on the NPY/AgRP neurones in the arcuate nucleus, which it is believed to get access to from the blood ( Batterham et al.
  • the peptide is released as PYY1-36, but a fraction - approximately 50 % - circulates as PYY3-36 which is a product of degradation by dipeptidylpeptidase-IV an enzyme which removes a dipeptide from the N-terminus of a peptide provided that a Pro or Ala is found in position two as in all three PP-fold peptides - PP, PYY and NPY ( Eberlein et al. 1989 Peptides 10: 797-803 ).
  • PYY in the circulation is a mixture of PYY1-36, which acts on both Y1 and Y2 receptors (as well as Y4 and Y5 with various affinities), and PYY3-36 - which has lower affinities for the Y1, Y4 and Y5 receptors than for the Y2 receptor.
  • PP is a hormone, which is released from endocrine cells in the pancreatic islets, almost exclusively governed by vagal cholinergic stimuli elicited by especially food intake ( Schwartz 1983 Gastroenterology 85:1411-25 ). PP has various effects on the gastrointestinal tract, but most of these are not observed in isolated cells and organs, and appear to be dependent on an intact vagal nerve supply ( Schwartz 1983 Gastroenterology 85:1411-25 ).
  • the PP receptors which are called Y4 receptors, are located in the brain stem with a strong expression in vagal motor neurones - activation of which results in the peripheral effects of PP - and in the nucleus tractus solitarirus (NTS) - activation of which results in the effects of PP as a satiety hormone ( Whitecomb et al. 1990 Am.J.Physiol. 259: G687-91 , Larsen & Kristensen 1997 Brain Res.Mol.Brain Res 48: 1-6 ). It should be noted that PP from the blood has access to this area of the brain since the blood brain barrier is "leaky" in this area where various hormones from the periphery are sensed.
  • PP acts through Y4 receptors for which it has a subnanomolar affinity as opposed to PYY and NPY which have nanomolar affinity for this receptor ( Michel et al. 1998 Pharmacol. Rev. 50: 143-150 ).
  • PP also has an appreciable affinity for the Y5 receptor, but it is not likely of physiological importance in relation to circulating PP due to both lack of access to the cells in the CNS where this receptor especially is expressed and due to the relatively low affinity for PP.
  • PP-fold peptide receptors There are four well established types of PP-fold peptide receptors in man: Y1, Y2, Y4, and Y5 which all recognize NPY and PYY with similar affinity.
  • Y3 receptor type which might prefer NPY over PYY, was suggested, but today this is not accepted as a real receptor subtype ( Michel et al. 1998 Pharmacol. Rev. 50: 143-150 ).
  • a Y6 receptor subtype has been cloned, however in man this is expressed in.a truncated form lacking TM-VII as well as the receptor tail and consequently at least on its own does not appear to form a functional receptor molecule.
  • Y1 receptors - affinity studies suggest Y1 binds NPY and PYY equally well and basically not PP.
  • Y2 receptors - affinity studies suggest Y2 binds NPY and PYY equally well and basically not PP.
  • Y4 receptors - affinity studies suggest that Y4 binds PP with subnanomolar affinity corresponding to the concentrations found in plasma whereas NPY and PYY are recognized with much lower affinity.
  • Y5 receptors - affinity studies suggest that Y5 binds NPY and PYY equally well, and also binds PP with lower affinity, which however is below the normal circulating levels of this hormone.
  • PYY3-36 is also recognized well by the Y5 receptor. However this receptor is to a large degree expressed in the CNS where PYY3-36 cannot get access to the receptor readily when administered in the periphery.
  • PP-fold peptides and analogs of these have been suggested for use in the treatment of obesity and associated diseases, including for example Prader Willi's syndrome, based on the demonstrated effects of certain of the these peptides in animal models and in man and on the fact that obese people have low basal levels of PYY and PP as well as lower meal responses of these peptides ( Holst JJ et al. 1983 Int.J.Obes. 7: 529-38 ; Batterham et al. 1990 Nature ). Infusion of PP in patients with Prader Willi's syndrome was early on shown to decrease food intake ( Berntson et al.
  • Y receptor PP-fold peptides or PP-fold peptide mimics which were specific for the selected Y receptor intended as target, and which stably preserve elements of the PP-fold structure important for receptor binding.
  • agents which are selective for the Y2 receptor over the Y1 and Y4 receptors.
  • the Y2 receptor is the receptor, which will give the beneficial effect on for example food intake and energy expenditure for the treatment of obesity, metabolic syndrome etc, and it is also the Y2 receptor which will: give the beneficial effect to obtain therapeutic angiogenesis in patients with for example peripheral vascular disease or coronary vascular disease.
  • an agent which acts as a Y2 receptor agonist is not particularly useful for such treatment unless it is selective for the Y2 receptor over the Y1 and the Y4 receptors.
  • Agonism on the Y1 receptor will, for example induce serious side effects in the cardiovascular system - increase in blood pressure - as well as renal system - natriuresis.
  • Y2 selectivity over the Y4 receptor is desirable, since the two natural Y2 and Y4 agonists, PYY and PP respectively, have many similar effects on for example the gastrointestinal tract - some of which could be beneficial - but some of which may cause unwanted side effects.
  • both Y2 and Y4 receptors promote anti-secretory effects in the small and large intestine through respectively a neuronal and a direct epithelial mode of action ( Cox et al. 2002 Br.J.Pharmacol. 135: 1505-12 ).
  • a neuronal and a direct epithelial mode of action Cox et al. 2002 Br.J.Pharmacol. 135: 1505-12 .
  • This invention relates to specific peptides which are highly selective for the Y2 receptor over the Y1 and Y4 receptors.
  • the notation hPP used herein refers to the human PP sequence, SEQ ID No:3.
  • the peptide [Lys4,Leu17,Ser30,Gln34]hPP has the human PP sequence SEQ ID No:3, but with lysine substituted at position 4, leucine substituted at position 17, serine substituted at position 30 and glutamine substituted at position 34.
  • the notation "oxidised Met” refers to a methionine residue wherein the sulfur atom of the side chain methylthio group is oxidised to a sulfoxide (one oxygen) or a sulfone (two oxygens).
  • the oxidised Met30 in peptide (iii) of the invention is a sulfoxide.
  • the three peptides and their analogues of the invention are Y receptor agonists which are highly selective for the Y2 receptor over the Y1 and Y4 receptors when measured by the affinity and/or potency assays described herein.
  • amino acids by their common names or abbreviations, such as valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg), aspartic acid (Asp), glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), tyrosine (Tyr), tryptophane (Trp), cysteine (Cys) and proline (Pro).
  • the amino acid in question is to be understood as the L-form.
  • the amino acid will be specifically referred to as such.
  • the L-form will be specified rather than inferred.
  • conservatively substituted denotes that one or more amino acids is replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. Non-limiting examples of conservative amino acid substitutions suitable for use in the present invention include those in the following Table and analogous substitutions of the original residue by non-natural alpha amino acids which have similar characteristics.
  • Conservatively substituted analogues of the invention may have, for example, up to 10 conservative substitutions, or in another embodiment up to 5, or in yet another embodiment 3 or fewer.
  • All three Y2 selective agonists with which the invention is concerned may be acylated at their N-terminus to confer resistance to aminopeptidase activity.
  • acylation may be with a carbon chain having from 2 to 24 carbon atoms, and N-terminal acetylation is a particular example.
  • modifications may be made to the three agonists of the invention, for the purpose of improving their pharmacokinetics, pharmacodynamics and metabolic properties.
  • modifications may involve linking the agonist to functional groupings (also known as motifs) known per se in the art of peptidic or proteinaceous pharmaceuticals.
  • functional groupings also known as motifs
  • Three particular modifications of particular benefit in the case of the agonists with which the invention is concerned, are linkage with serum albumin binding motifs, or glycosaminoglycan (GAG) binding motifs, or PEGylation.
  • Serum albumin binding motifs are typically lipophilic groups, incorporated to enable a prolonged residence in the body upon administration or for other reasons, which may be coupled in various known ways to peptidic or proteinaceous molecules, for example i) via a covalent linkage to e.g. a functional group present on a side-chain amino acid residue, ii) via a functional group inserted in the peptide or in a suitable derivatized peptide, iii) as an integrated part of the peptide.
  • WO 96/29344 Novo Nordisk A/S
  • P. Kurtzhals et al. 1995 Biochemical J. 312: 725-31 and L.B.Knudsen et al. 2000 J.Med.Chem. 43: 1664-69 describe a number of suitable lipophilic modifications which can be employed in the case of the agonists with which this invention is concerned.
  • Suitable lipophilic groups include optionally substituted, saturated or unsaturated, straight or branched hydrocarbon groups of from 10 to 24 carbon atoms. Such groups may form, or may form part of, a side chain to the backbone of the agonist, for example by ether, thioether, amino, ester or amide linkage to a side chain of an amino acid residue in the backbone, or to a backbone carbon or a branch from a backbone carbon of a non-peptidic linker radical in the backbone of a PP-fold mimic agonist.
  • the chemistry strategy for attachment of the lipophilic group is not critical, but the following side chains including lipophilic groups are examples which can be linked to backbone carbon of the agonist, or suitable branch therefrom:
  • the lipophilic group-containing side chain is a C 12 , C 14 , C 16 or C 18 acyl group, for example a tetradecanoyl group, acylating an amino group present in the side chain of a residue of the backbone of the agonist.
  • modified agonists for use in accordance to provide improved serum binding characteristics is a strategy which may be applied in general, and particularly in the case of the specific agonists listed above.
  • suitable modified agonists include [Lys4,N-(N'-tetradecanoyl)-gammagluatamoyl-Lys13,Leu17,Ser30,Gln34]PP or [Lys4,N-(N'-hexadecanoyl)-gammagluatamoyl-Lys13,Leu17,Thr30,Gln34]PP and conservatively substituted analogues thereof.
  • the agonists with which this invention are concerned may be modified by incorporation of the GAG binding motif as, or as part of, a side chain to the backbone of the agonist.
  • GAG-binding motifs for incorporation in this way include the amino acid sequences XBBXBX and/or XBBBXXBX, wherein B is a basic amino acid residue and X is any amino acid residue.
  • a plurality, for example three, of such sequences may be incorporated in a concatameric (straight chain) or dendrimeric (branched chain) fashion.
  • Specific concatameric GAG motifs include Ala-Arg-Arg-Arg-Ala-Ala-Arg-Ala-Ala-Arg-Arg-Arg-Ala-Ala-Arg-Ala, and Ala-Arg-Arg-Arg-Ala-Ala-Arg-Ala-Ala-Arg-Arg-Arg-Ala-Ala-Arg-Arg-Ala-Ala-Arg-Ala (both of which may, for example be coupled through an amide bond formed between the C-terminus of the concatameric GAG-binding motif and an amino group in the side chain of a backbone amino acid of the agonist, such as the epsilon amino group of Lys13 in the agonist [Lys4,Lys13,Leu17,Ser30,Gln34]PP or [Lys4,Lys13,Leu17,Thr30,Gln34]PP.
  • the GAG motif may be covalently linked to the C- or (preferably) N-terminus of the agonist, either directly or via a linker radical.
  • the GAG-binding motif may comprise the amino acid sequence XBBXBX and/or XBBBXXBX, wherein B is a basic amino acid residue and X is any amino acid residue, for example the sequence [XBBBXXBX] n where n is 1 to 5, B is a basic amino acid residue and X is any amino acid residue.
  • the Y2 selective agonists with which the present invention is concerned are useful, inter alia, for therapeutic angiogenesis.
  • the agonists preferably comprise a glycosaminoglycan (GAG) binding motif as discussed above.
  • GAG glycosaminoglycan
  • Such motifs ensure that the agonists bind to GAGs in the extracellular matrix, and thereby ensures prolonged local exposure of the Y2 receptors in that tissue.
  • Growth factors, chemokines etc bind to GAGs through patches of basic amino acids, which interact with the acidic sugars of the GAGs.
  • sequences are placed for example in a concatameric or dendrimeric construct where for example three such sequences are presented -for example each as a ARRRAARA sequence - the resulting 24-mer peptide - for example ARRRAARA-ARRRAARA-ARRRAARA - ensures a retention in the extracellular matrix similar to high molecular weight polylysine, i.e. it is not washed out during a 4 hour perfusion period ( Sakharov et al. FEBS Lett 2003, 27: 6-10 ).
  • Growth factors and chemokines are naturally constructed with two types of binding motifs: one binding motif for the receptor through which signal transduction is achieved and one binding motif for GAG's through which attachment, and long-lasting local activity is achieved.
  • Peptides such as PYY and NPY are neuropeptides and hormones, which are rather rapidly washed out of the tissue and are not optimized for long-lasting local activity.
  • a bi-functional molecule similar to the growth factors and chemokines is constructed having both a receptor binding epitope in the PP-fold peptide part and a GAG-binding motif.
  • An example of such an agonist is [Lys4,N- ⁇ (Ala-Arg-Arg-Arg-Ala-Ala-Ala-Arg-Ala) 3 ⁇ -Lys13,Leu17,Ser30,Gln34]PP.
  • a polyalkyleneoxide radical or radicals is/are covalently coupled to peptidic or proteinaceous drugs to improve effective half life in the body following administration and to reduce immunogenicity, increase solubility etc.
  • the term derives from the preferred polyalkyleneoxide used in such processes, namely that derived from ethylene glycol - polyethyleneglycol, or "PEG".
  • a suitable PEG radical may be attached to the agonist by any convenient chemistry, for example via a backbone amino acid residue of the agonist, and may also incorporate cleavable linkers ( FEBS Lett. 2005 Apr 25;579(11):2439-44 .).
  • a frequently used attachment group is the epsilon-amino group of lysine or the N-terminal amino group.
  • Other attachment groups include a free carboxylic acid group (e.g. that of the C-terminal amino acid residue or of an aspartic acid or glutamic acid residue), suitably activated carbonyl groups, mercapto groups (e.g.
  • cysteine residue aromatic acid residues (e.g. Phe, Tyr, Trp), hydroxy groups (e.g. that of Ser, Thr or OH-Lys), guanidine (e.g. Arg), imidazole (e.g. His), and oxidized carbohydrate moieties.
  • aromatic acid residues e.g. Phe, Tyr, Trp
  • hydroxy groups e.g. that of Ser, Thr or OH-Lys
  • guanidine e.g. Arg
  • imidazole e.g. His
  • oxidized carbohydrate moieties oxidized carbohydrate moieties.
  • the agonist when the agonist is PEGylated it usually comprises from 1 to 5 polyethylene glycol (PEG) molecules such as, e.g. 1, 2 or 3 PEG molecules.
  • PEG polyethylene glycol
  • Each PEG molecule may have a molecular weight of from about 5 kDa (kiloDalton) to about 100 kDa, such as a molecular weight of from about 10 kDa to about 40 kDa, e.g., about 12 kDa or in another embodiment no more than about 20 kDa.
  • PEG 40 kDa is the PEGylating agent.
  • Suitable PEG molecules are available from Shearwater Polymers, Inc. and Enzon, Inc. and may be selected from SS-PEG, NPC-PEG, aldehyde-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC, SC-PEG, tresylated mPEG ( US 5,880,255 ), or oxycarbonyl-oxy-N-dicarboxyimide-PEG ( US 5,122,614 ).
  • PEGylated agonists of the invention are [Lys4,N-PEG5000-Lys13,Leu17,Ser30,Gln34]PP and [Lys4,N-PEG5000-Lys13,Leu17,Thr30,Gln34]PP.
  • Serum albumin, GAG and PEG Serum albumin, GAG and PEG
  • the modification to the agonist is attachment of a group to facilitate serum binding, GAG binding or improved stability via PEGylation
  • the serum albumin binding motif or GAG binding motif, or PEG radical may be, or may form part of, a side chain of a backbone carbon of the agonist corresponding to any of the following positions: 1, 3, 6, 7, 10, 11, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 28, 29, and 32.
  • the selective Y2 receptor agonists may be used as fusion proteins where they are linked for example to albumin or another protein or carrier molecule which provides beneficial pharmacokinetic or other types of properties such as for example decreased renal elimination.
  • albumin or another protein or carrier molecule which provides beneficial pharmacokinetic or other types of properties such as for example decreased renal elimination.
  • linkers which can be used for such a covalent attachment as known in the art, just as there are multiple proteins or carriers which can be used.
  • covalent attachment of the selective Y2 peptide agonist to albumin is preferred, and at the positions in peptides which have been pointed out elsewhere herein in relation to modifications with the various motifs.
  • a peptide is fused to the C-terminal end of a large biomolecule such as albumin.
  • fusion proteins can be produced through various semisynthetic techniques where the peptide may be made through peptide synthesis as described herein and the biomolecule through recombinant technology.
  • the fusion protein may also be made entirely as a recombinant molecule expressed for example as a precursor molecule extended by a Gly-Lys-Arg sequence which when expressed as a secretory protein in eukaryotic cells will be cleaved by biosynthetic enzymes and the Gly turned into the carboxyamide on the C.terminal Tyr residue of the C-terminal Y2 receptor recognition sequence.
  • Another stabilising modification involves the covalent attachment of a stabilizing peptide sequence of 4-20 amino acid residues covalently at the N-and/or the C-terminus, preferably the N-terminus.
  • the amino acid residues in such a peptide are selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and the like.
  • N-terminal peptide attachment comprises 4, 5 or 6 Lys residues, for example Lys-Lys-Lys-Lys-Lys-Lys-[Lys4,Leu17,Ser30,Gln34]PP. These can be linked at the N-terminus of the peptide agonist.
  • Lys-Lys-Lys-Lys-Lys-Lys-[Lys4,Leu17,Ser30,Gln34]PP can be linked at the N-terminus of the peptide agonist.
  • a general description of such stabilizing peptide extensions is given in WO 99/46283 (Zealand Pharmaceuticals), which is hereby incorporated by reference.
  • the receptor agonists with which the invention is concerned may be prepared by well-known methods such as, e.g., a synthetic, semisynthetic and/or recombinant method.
  • the methods include standard peptide preparation techniques such as, e.g., solution synthesis, and solid-phase synthesis. Based on textbook and general knowledge within the field, a person skilled in the art knows how to proceed in order to obtain the agonists and derivatives or modifications thereof.
  • the Y2-specific agonists with which the invention is concerned are of value in the treatment of conditions responsive to activation of Y2 receptors.
  • the peptides of the invention may be used in the preparation of a medicament for treatment of cardiovascular disease, hypertension, atherosclerosis, coronary artery disease, peripheral vascular disease, coronary vascular disease, myocardial infarction, stroke, atherosclerosis, or thromboembolic disease, hypercholesterolemia, hyperlipidemia or sleep apnea.
  • NPY may act as an angiogenic factor (Zukowska- Grojec et al. 1998 Circ.Res. 83: 187-95 ).
  • NPY is a potent angiogenic factor which gives rise to vascular tree-like structures showing vasodilation as observed otherwise only with fibroblast growth factor-2 (FGF-2) and not for example vascular endothelial growth factor (VEGF) angiogenic structures ( Ekstrand et al. 2003 PNAS 100: 6033-38 ).
  • FGF-2 fibroblast growth factor-2
  • VEGF vascular endothelial growth factor
  • induction of angiogenesis would be beneficial in peripheral vessels as well as in coronary vessels.
  • induction of angiogenesis is believed to be beneficial for securing reperfusion after myocardial infarction.
  • FGF-2 has been proposed to be an especially efficient agent for induction of angiogenesis in patients with cardiovascular diseases.
  • FGF-2 is a growth factor and has the potential of stimulating tumor growth also by providing angiogenesis.
  • NPY acting through Y2 receptors induces neovascularization of a similar type as induced by FGF-2.
  • NPY is a neuropeptide and not a classical growth factor and has not been implicated in inducing tumor growth.
  • a Y2 agonist is a useful agent for therapeutic angiogenesis.
  • the agonist does not show Y1 receptor agonism because this will give unwanted cardiovascular effects.
  • all the peptides with which the invention is concerned are Y2 selective receptor agonists and are especially useful therapeutic agents also with respect of inducing angiogenesis. They are particularly useful when modified with GAG binding motifs, as discussed above.
  • GAG glycosaminoglycans
  • the peptides incorporate one or more GAG binding motifs, which ensures that they attach to GAGs in the extracellular matrix to induce optimal angiogenesis after administration.
  • This can, for example, be by intravenous or intraarterial administration or, for example, direct administration into the coronary arteries in order to induce cardiac angiogenesis during coronary artery disease and/or post acute myocardial infarction.
  • Such a compound can be administered through intraarterial injection in the femoral artery for treatment of peripheral vascular disease. It can also be, for example, by topical local administration to skin lesions in order to promote improved wound healing.
  • a prolonged Y receptor exposure efficient in inducing angiogenesis can also be obtained by using a peptide according to the present invention modified with a serum albumin binding motif.
  • the Y2 selective agonists comprise a GAG-binding motif, which is placed in a position where it does not impair the stability of the peptide or impair the potency and selectivity of the peptide.
  • the invention relates to the use the Y2 selective receptor agonist for inducing angiogenesis associated with diseases or conditions such as e.g., cardiovascular diseases including peripheral vascular disease with symptoms such as cladicatio intermittens, coronary artery disease and myocardial infarction,
  • the therapeutically effective amount of a Y2 receptor agonist according to the invention will be dependent on specific agonist employed, the age, weight and condition of subject being treated, the severity and type of the condition or disease being treated, the manner of administration and the strength of the composition applied.
  • a therapeutically effective amount of the Y2 receptor agonist thereof can vary from about 0.01 ⁇ g per kilogram (kg) body weight to about 1 g per kg body weight, such as about 1 ⁇ g to about 5 mg per kg body weight, or about 5 ⁇ g to about 1 mg per kg body weight.
  • the receptor agonist is administered to a subject at 0.5 to 135 picomole (pmol) per kg body weight, or about 72 pmol per kg body weight.
  • nmol is administered as a subcutaneous injection, such as from about 2 to about 20 nmol, or about 1.0 nmol is administered as a subcutaneous injection.
  • the exact dose is readily determined by one skilled in the art based on the potency of the specific compound (such as the receptor agonist) utilized, the age, weight, sex and physiological condition of the subject.
  • the amounts can be divided into one or several doses for administration daily, every second day, weekly, every two weeks, monthly or with any other suitable frequency. Normally, the administration is once or twice daily.
  • the Y2 receptor agonist as well as cosmetic or pharmaceutical compositions according to the invention can be administered by any route, including the enteral (e.g. oral administration) or parenteral route.
  • the parenteral route is preferred and includes intravenous, intraarticular, intraperitoneal, subcutaneous, intramuscular, intrasternal injection and infusion as well as administration by the sublingual, transdermal, topical, transmucosal including nasal route, or by inhalation such as, e.g., pulmonary inhalation.
  • the subcutaneous and/or the nasal administration route is preferred.
  • the receptor agonists can be administered as such dispersed in a suitable vehicle or they can be administered in the form of a suitable pharmaceutical or cosmetic composition. Such compositions are also within the scope of the invention. In the following are described suitable pharmaceutical compositions. A person skilled in the art will know how that such composition may also be suitable for cosmetic use or he will know how to adjust the compositions to cosmetic compositions by use of suitable cosmetically acceptable excipients.
  • receptor agonists also denoted “compounds”
  • compounds for use in medicine or cosmetics are normally presented in the form of a pharmaceutical composition comprising the specific compound or a derivative thereof together with one or more physiologically or pharmaceutically acceptable excipients.
  • the compounds may be administered to an animal including a mammal such as, e.g., a human by any convenient administration route such as, e.g., the oral, buccal, nasal, ocular, pulmonary, topical, transdermal, vaginal, rectal, ocular, parenteral (including inter alia subcutaneous, intramuscular, and intravenous cf. above), route in a dose that is effective for the individual purposes.
  • parenteral administration route is preferred.
  • the receptor agonists are administered subcutaneously and/or nasally. It is well known in the art that subcutaneous injections can be easily self-administered.
  • composition suitable for a specific administration route is easily determined by a medical practitioner for each patient individually.
  • Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin.
  • the pharmaceutical composition comprising a compound according to the invention may be in the form of a solid, semi-solid or fluid composition.
  • the composition is normally in the form of a fluid composition or in the form of a semi-solid or solid form for implantation.
  • Fluid compositions which are sterile solutions or dispersions can utilized by for example intravenous, intramuscular, intrathecal, epidural, intraperitoneal or subcutaneous injection of infusion.
  • the compounds may also be prepared as a sterile solid composition, which may be dissolved or dispersed before or at the time of administration using e.g. sterile water, saline or other appropriate sterile injectable medium.
  • the fluid form of the composition may be a solution, an emulsion including nano-emulsions, a suspension, a dispersion, a liposomal composition, a mixture, a spray, or a aerosol (the two latter types are especially relevant for nasal administration).
  • Suitable mediums for solutions or dispersions are normally based on water or pharmaceutically acceptable solvents e.g. like an oil (e.g. sesame or peanut oil) or an organic solvent like e.g. propanol or isopropanol.
  • a composition according to the invention may comprise further pharmaceutically acceptable excipients such as, e.g., pH adjusting agents, osmotically active agents e.g. in order to adjust the isotonicity of the composition to physiologically acceptable levels, viscosity adjusting agents, suspending agents, emulsifiers, stabilizers, preservatives, antioxidants etc.
  • a preferred medium is water.
  • compositions for nasal administration may also contain suitable non-irritating vehicles such as, e.g., polyethylene glycols, glycofurol, etc. as well as absorption enhancers well known by a person skilled in the art (e.g. with reference to Remington's Pharmaceutical Science)
  • suitable non-irritating vehicles such as, e.g., polyethylene glycols, glycofurol, etc. as well as absorption enhancers well known by a person skilled in the art (e.g. with reference to Remington's Pharmaceutical Science)
  • the receptor agonists can be formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable excipient or carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the composition.
  • a pharmaceutically acceptable excipient or carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the composition.
  • the formulations are prepared by contacting the receptor agonist uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. Due to the amphiphatic nature of the peptides described herein suitable forms also include micellar formulations, liposomes and other types of formulations comprising one or more suitable lipids such as, e.g., phospholipids and the like.
  • aqueous carriers Preferably, they are suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.
  • buffer substances include acetate, citrate, phosphate, borate, carbonate such as, e.g., sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers.
  • compositions may also be designed to controlled or prolonged delivery of the receptor agonist after administration in order to obtain a less frequent administration regimen. Normally a dosage regimen including 1-2 daily administrations is considered suitable, but within the scope of the present invention is also included other administration regimens such as, e.g., more frequent and less frequent.
  • a suitable vehicle including e.g. lipids or oils may be employed in order to form a depot at the administration site from which the receptor agonist is slowly released into the circulatory system, or an implant may be used.
  • Suitable compositions in this respect include liposomes and biodegradable particles into which the receptor agonist has been incorporated.
  • the solid composition may be in the form of tablets such as, e.g. conventional tablets, effervescent tablets, coated tablets, melt tablets or sublingual tablets, pellets, powders, granules, granulates, particulate material, solid dispersions or solid solutions.
  • tablets such as, e.g. conventional tablets, effervescent tablets, coated tablets, melt tablets or sublingual tablets, pellets, powders, granules, granulates, particulate material, solid dispersions or solid solutions.
  • a semi-solid form of the composition may be a chewing gum, an ointment, a cream, a liniment, a paste, a gel or a hydrogel.
  • suitable dosages forms of the pharmaceutical compositions according to the invention may be vagitories, suppositories, plasters, patches, tablets, capsules sachets, troches, devices etc.
  • the dosage form may be designed to release the compound freely or in a controlled manner e.g. with respect to tablets by suitable coatings.
  • the pharmaceutical composition may comprise a therapeutically effective amount of a compound according to the invention.
  • the content of a compound of the invention in a pharmaceutical composition of the invention is e.g. from about 0.1 to about 100% w/w of the pharmaceutical composition.
  • compositions may be prepared by any of the method well known to a person skilled in pharmaceutical formulation.
  • the compounds are normally combined with a pharmaceutical excipient, i.e. a therapeutically inert substance or carrier.
  • the carrier may take a wide variety of forms depending on the desired dosage form and administration route.
  • the pharmaceutically acceptable excipients may be e.g. fillers, binders, disintegrants, diluents, glidants, solvents, emulsifying agents, suspending agents, stabilizers, enhancers, flavours, colors, pH adjusting agents, retarding agents, wetting agents, surface active agents, preservatives, antioxidants etc. Details can be found in pharmaceutical handbooks such as, e.g., Remington's Pharmaceutical Science or Pharmaceutical Excipient Handbook.
  • the peptides were synthesized with an Applied Biosystem Inc. (ABI) Model 433 automated synthesizer based on the solid phase peptide synthesis (SPPS) approach using Fmoc chemistry. All the reagents for the ABI synthesizer were purchased from ABI (except piperidine was from Aldrich). Fmoc amino acids were purchased from ABI. Rink Amide MBHA resins were from Novabiochem. Standard 0.25 mmole FastMoc chemistry was used.
  • the general Fmoc chemistry protocol for SPPS includes: 1) cleavage of the Fmoc protection groups with 20% piperidine; 2).
  • HBTU 2-(1 H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
  • the activated Fmoc amino acid was formed almost instantaneously and the solution was transferred directly to the reaction vessel.
  • the step of Fmoc deprotection was monitored and controlled by conductivity measurement.
  • the final synthesis was product was washed extensively with NMP and dichloromethane (DCM).
  • the peptide solution was separated from the resin by filtration and precipitated in 40 ml of cold diethyl ether.
  • the peptide was recovered by centrifugation and washed 2 x 40 ml of cold diethyl ether.
  • the peptide was lyophilized and stored at -20°C before purification.
  • the peptide powder was dissolved in 50% acetic acid solution and injected onto a semi-preparative reverse phase HPLC column for purification.
  • a HPLC system with dual wavelength (220 nm and 280 nm) uv detector was used.
  • a linear gradient of acetonitrile was programmed and introduced to the column to separate the peptide product from other substances.
  • the eluant was collected by a fraction collector, and the individual separation fractions were subjected to both analytical HPLC and MALDI-TOF MS for characterization to ensure identity and purity.
  • the resin was air-dried and transferred into a glass vial and 10 ml of freshly prepared cleavage reagent (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS) in water) was added.
  • TSA trifluoroacetic acid
  • TIS triisopropylsilane
  • the deprotection reaction was carried out for 4 hours at room temperature with constant stirring.
  • the supernatant was then separated from the resin by filtration.
  • the peptide was precipitated with 40 ml of ice-cold diethyl ether followed by centrifugation (6 min at 3,500 x rpm) for recovery.
  • the precipitated peptide was washed twice with cold diethyl ether.
  • the peptide solution was freeze-dried overnight.
  • the peptide was re-dissolved in 50% acetic acid and purified on a Vydac C8 reverse phase HPLC column (1.0 cm I.D., 25 cm length with 5 ⁇ m particle size, and 300 A pore size) using a linear gradient of 0-70% solvent B with solvent A in 70 min at a flow rate 3 ml/min.
  • the composition of solvents A and B were as follows: A: 0.1% TFA, 2% acetonitrile in water; B: 0.1% TFA in 95% aqueous acetonitrile.
  • the fractions were collected at every 0.1 min. Aliquots of each fraction were analyzed by both MS and analytical RP-HPLC. The fractions that contained a single u.v.
  • [Lys4, Leu17, Met30, Gln34]hPP was prepared according to Example 1, with the exception that Fmoc-Met was used instead of Fmoc-Thr-OtBu.
  • the purified [Lys4, Leu17, Met30, Gln34]hPP peptide was then oxidized in 0.14% hydrogen peroxide to yield [Lys4, Leu17, Met-sulfoxide30, Gln34]hPP: 0.65 mg of [Lys4, Leu17, Met30, Gln34]hPP was dissolved in 1.5 ml of phosphate buffer. 75 ⁇ l of 3 % hydrogen peroxide was then added to the peptide solution. The oxidation reaction was carried out for 12 hours in the dark.
  • the coding region of the rhesus monkey NPY- Receptors (Y1, Y2, Y4) were subcloned into the pcDNA3.1/Zeo vector containing HA-signal sequence-Flag Tag on the amino terminus as described in X.-M. Guan et. al. J. Biol. Chem. 267(31):21995-21998(1992 ), and used for expression, binding, and functional studies to identify Y2-selective peptides.
  • the rhY1 is identical to Genbank sequence AF303089.
  • the rhY2 is identical to Genbank sequence AF303090.
  • the rhY4 is identical to Genbank sequence AY149475.1.
  • Affinity of test compounds for the rhesus monkey Y2 receptor is determined in a competition binding assay using human 125I-PYY binding in CHO cells stably transfected with the rhesus monkey Y2 receptor.
  • the stable transfected CHO cells are transferred to 48-well culture plates one day prior assay at a density of 2,500 cells per well aiming at 5 - 8 % binding of the radioactive ligand.
  • competition binding experiments are performed for 3 hours at 4 C° using 12 pM of human 125I-PYY (Amersham, Little Chalfont, UK). Binding assays are performed in 0.5 ml of a 50 mM Hepes buffer, pH 7.4, supplemented with 1 mM CaCl2, 5 mM MgCl2, and 0.1 % (w/v) bovine serum albumin and 100 ⁇ g/ml bacitracin.
  • Non-specific binding is determined as the binding in the presence of 1 ⁇ M of unlabeled human PYY.
  • Cells are washed twice in 0.5 ml of ice-cold buffer and 0.5-1 ml of lysis buffer (8 M Urea, 2 % NP40 in 3 M acetic acid) is added and the bound radioactivity is counted in a gamma counter. Determinations are made in triplicates. Steady state binding is reached with the radioactive ligand under these conditions.
  • IC50 values were calculated using a standard pharmacological data handling software, Prism 3.0 (graphPad Sofware, San Diego, USA).
  • Protocol as for the Y2 affinity assay except that CHO cells stably expressing rhesus monkey Y4 are used and cells are transferred to culture plates at a density of 125,000 cells per well.
  • the competition assay uses human 125I-PP, and human PP is used for the determination of non-specific binding.
  • Protocol as for the Y2 affinity assay except that CHO cells stably expressing rhesus monkey Y1 are used and cells are transferred to culture plates at a density of 23,000 cells per well.
  • the competition assay uses human 125I-PYY, and human PYY is used for the determination of non-specific binding.
  • Table 1 Compound Competition binding w 128 I-PYY at Y2 (Ki, nM) SEQ ID No: 2 PYY 0.09 (8) SEQ ID No: 1 NPY 0.11 (1) PYY3-36 0.32 (3) SEQ ID No: 3 PP >1000 SEQ ID No: 4 [Lys4,Leu17, Ser30,Gln34]-PP 0.51 (2) SEQ ID No: 5 [Lys4, Leu17, Thr30, Gln34]-PP 0.47 (4) SEQ ID No: 6 [Lys4, Leu17, Met(O)30,Gln34]-PP 0.06 (1) Values in parentheses shows number of independent experiments.
  • the peptides represented by SEQ ID Nos. 4-6 are ⁇ 100-fold selective against the Y1 receptor and ⁇ 20-fold selective against the Y4 receptor.
  • Potency of the test compounds on the rhesus monkey Y2 receptor is determined by performing dose-response experiments in COS-7 cells transiently transfected with the rhesus monkey Y2 receptor as well as a chimeric G protein, Gqi5 which ensures that the Y2 receptor couples through a Gq pathway leading to an increase in inositol phosphate turnover.
  • COS-7 cells are transferred to 96-wells culture plates at a density of 30,000 cells per well and incubated for 24 hours with 0.5 ⁇ Ci of [3H]-myo-inositol (Amersham, PT6-271) in 100 ⁇ l medium supplemented with 10% fetal calf serum, 2 mM glutamine and 0.01 mg/ml gentamicin per well.
  • Cells are washed twice in buffer, 20 mM HEPES, pH 7.4, supplemented with 140 mM NaCl, 5 mM KCI, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose, 0.05 % (w/v) bovine serum; and are incubated in 100 ⁇ l buffer supplemented with 10 mM LiCl at 37°C for 30 min. After stimulation with various concentrations of peptide for 45 min at 37°C, cells are extracted with 50 ⁇ l 10 % ice-cold perchloric acid followed by incubation on ice for 30 min. 20 ⁇ l of the perchloric acid cell solution is transferred into a solid white 96 wells plate.
  • Protocol as for the Y2 potency assay except that rhesus monkey Y4-transformed COS-7 cells are used.
  • Protocol as for the Y2 potency assay except that rhesus monkey Y1-transformed COS-7 cells are used.
  • Table 2 Compound IP3 EC50 values at Y2 (nM) SEQ ID No: 2 PYY 0.18 (9) SEQ ID No: 1 NPY 0.60 (4) SEQ ID No: 3 PP > 1 ⁇ M (2) PYY3-36 0.29 (9) SEQ ID No: 3 [Lys4,Leu17, Ser30,Gln34]-PP 0.67 (3) SEQ ID No: 4 [Lys4, Leu17, Thr30, Gln34]-PP 0.71 (4) SEQ ID No: 5 [Lys4, Leu17, Met(O)30,Gln34]-PP 0.50 (2) Values in parentheses shows number of independent experiments.
  • the peptides represented by SEQ ID Nos. 4-6 are ⁇ 100-fold selective against the Y1 receptor and ⁇ 20-fold selective against the Y4 receptor.
  • test compounds to bind to GAGs is monitored in an in vitro assay using immobilized heparin, i.e. for example either a HiTrap heparin-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) or a heparin HPLC columns which are eluted with a 50-min linear gradient of 0-0.5 M NaCl in 50 mM sodium phosphate (pH 7.3) containing 2 mM DTT and 1 mM MgEDTA at a flow rate of 1 ml/min. For regeneration, the column was washed with 1 M NaCl in buffer A from 51-55 min. For initial analytical purposes a step-gradient of NaCl can be used.
  • immobilized heparin i.e. for example either a HiTrap heparin-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) or a heparin HPLC columns which are eluted with a

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Claims (11)

  1. Peptide sélectionné dans le groupe composé de :
    (i) [Lys4,Leu17,Ser30,Gln34]hPP (SEQ ID n° 4) ;
    (ii) [Lys4,Leu17,Thr30,Gln34]hPP (SEQ ID n° 5) ;
    (iii) [Lys4,Leu17,Met30 oxydée,Gln34]hPP (SEQ ID n° 6) ;
    et de modifications de (i), (ii) ou (iii), lesdites modifications se composant de (a) substitutions conservatives dans une ou plusieurs positions autres que les positions 4, 17, 30 et 34 de (i), (ii) et (iii) et/ou (b) une acylation N-terminale de (i), (ii) ou (iii), une PEGylation de (i), (ii) ou (iii) ou un couplage covalent de (i), (ii) ou (iii) à un motif de liaison à la sérumalbumine, un motif de liaison au glycosaminoglycane ou un motif induisant une hélice, ledit couplage covalent se faisant à un résidu du peptide (i), (ii) ou (iii) ou à un résidu substitué de manière conservative du peptide (i), (ii) ou (iii) dans une ou plusieurs positions autres que les positions 4, 17, 30 et 34, et qui fournit un groupe fonctionnel pour une telle liaison covalente.
  2. Peptide tel que revendiqué dans la revendication 1, dans lequel la Met30 oxydée est un sulfoxyde.
  3. Peptide tel que revendiqué dans la revendication 1 ou la revendication 2, qui est acylé au niveau de son extrémité N-terminale.
  4. Peptide tel que revendiqué dans l'une quelconque des revendications précédentes, qui comprend un motif de liaison à la sérumalbumine ou un motif de liaison à un glycosaminoglycane (GAG) ou un motif induisant une hélice, ou est PEGylé.
  5. Peptide tel que revendiqué dans la revendication 4, dans lequel le motif de liaison à la sérumalbumine comprend un groupe hydrocarbure ramifié ou linéaire, saturé ou non saturé, éventuellement substitué, comportant 10 à 24 atomes de carbone.
  6. Peptide tel que revendiqué dans la revendication 4, dans lequel le motif de liaison au GAG est une séquence d'acides aminés qui est, ou fait partie d'une chaîne latérale du squelette du peptide.
  7. Peptide tel que revendiqué dans la revendication 6, dans lequel le motif de liaison au GAG comprend la séquence d'acides aminés XBBXBX et/ou XBBBXXBX, où B est un résidu d'acide aminé basique et X est un résidu d'acide aminé quelconque.
  8. Peptide tel que revendiqué dans la revendication 4, dans lequel le PEG est un polyéthylèneglycol ou un oxyde de polyéthylène ayant un poids moléculaire au plus égal à 20 kDa.
  9. Peptide tel que revendiqué dans la revendication 4, dans lequel le peptide induisant l'hélice a 4-20 résidus d'acides aminés sélectionnés dans le groupe composé de Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, et des résidus d'acides aminés de formule -NH-C(R1)(R2)-CO-, dans laquelle R1 est l'hydrogène et R2 est un groupe alkyle en C1-C6 éventuellement substitué, phényle ou phénylméthyle, ou R1 et R2, pris conjointement avec l'atome de C auquel ils sont liés, forment un cycle cyclopentyle, cyclohexyle ou cycloheptyle.
  10. Utilisation d'un peptide tel que revendiqué dans l'une quelconque des revendications précédentes dans la préparation d'un médicament destiné au traitement d'une maladie cardiovasculaire, de l'hypertension, de l'athérosclérose, d'une maladie des artères coronaires, d'une maladie vasculaire périphérique, d'une maladie vasculaire coronaire, d'un infarctus du myocarde, d'un AVC ou d'une maladie thromboembolique, d'une hypercholestérolémie, d'une hyperlipidémie ou d'une apnée du sommeil.
  11. Composition pharmaceutique comprenant un ou plusieurs peptides tels que revendiqués dans l'une quelconque des revendications 1 à 9 avec un excipient acceptable sur le plan pharmaceutique.
EP05794808A 2005-09-21 2005-09-21 Agonistes selectifs du recepteur y2 pour applications therapeutiques Not-in-force EP1934245B1 (fr)

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US8871758B2 (en) 2011-03-08 2014-10-28 Sanofi Tetrasubstituted oxathiazine derivatives, method for producing them, their use as medicine and drug containing said derivatives and the use thereof
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EP2683704B1 (fr) 2011-03-08 2014-12-17 Sanofi Dérivés oxathiazine ramifiés, procédé pour leur préparation, utilisation en tant que médicament, agents pharmaceutiques contenant ces dérivés et leur utilisation
EP2567959B1 (fr) 2011-09-12 2014-04-16 Sanofi Dérivés d'amide d'acide 6-(4-hydroxy-phényl)-3-styryl-1h-pyrazolo[3,4-b]pyridine-4-carboxylique en tant qu'inhibiteurs de kinase
RU2641198C3 (ru) 2012-03-22 2021-12-10 Ново Нордиск А/С Композиции glp-1 пептидов и их получение
JP6202669B2 (ja) 2013-04-26 2017-09-27 国立研究開発法人国立循環器病研究センター ペプチド及びその複合体、組織修復用スキャフォールド及びその表面処理方法、並びに表面処理液又は処理液のセット
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NO20081910L (no) 2008-06-20
CN101268096A (zh) 2008-09-17
CA2623094A1 (fr) 2007-04-12
WO2007038943A1 (fr) 2007-04-12
US20080255046A1 (en) 2008-10-16
AU2005337101A1 (en) 2007-04-12
BRPI0520566A2 (pt) 2009-05-19
IL189948A0 (en) 2008-08-07
ATE516301T1 (de) 2011-07-15
JP2009508886A (ja) 2009-03-05
EA200800875A1 (ru) 2008-08-29
US7851590B2 (en) 2010-12-14

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